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Acta Biochim Biophys |
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doi:10.1111/j.1745-7270.2006.00166.x |
Nuclear Factor-kB Signaling Pathway Constitutively
Activated in Esophageal Squamous Cell Carcinoma Cell Lines and Inhibition of Growth
of Cells by Small Interfering RNA
Fang TIAN, Wei-Dong ZANG,
Wei-Hong HOU, Hong-Tao LIU, and Le-Xun XUE*
Laboratory for Cell Biology,
Medical College, Zhengzhou University, Zhengzhou 450052, China
Received: January 11, 2006
Accepted: February 27, 2006
This study was supported by a grant from the “211 Project”
(Education Ministry of China 2002-2)
*Corresponding
author: Tel, 86-371-66658332; Fax, 86-371-66997182; E-mail,
[email protected]
Abstract Although constitutive nuclear
factor (NF)-kB activation has been reported
in many human tumors, the role of the NF-kB
pathway in esophageal squamous cell carcinoma (ESCC) has not been known. In
this study, NF-kB pathway in two ESCC cell
lines was investigated using immunocytochemistry, Western blot and reverse
transcription-polymerase chain reaction. The activation of NF-kB DNA binding was determined by electrophoretic
mobility-shift assay. RNA interference was used to specifically inhibit the
expression of p65. Growth of cells was evaluated by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The results
showed that p50, p65, IkBa,
p-IkBa
and IkB kinase b were expressed and mainly localized in
the cytoplasm. Reverse transcription-polymerase chain reaction results showed
the constitutive expressions of p50, p65 and IkBa mRNA in the two ESCC cell lines.
Furthermore, the nuclear extracts revealed that p50 and p65 translocated to the
nucleus had DNA-binding activity. Finally, small interfering RNA of p65
decreased the expression of p65, and the viability of cells transfected with
p65 small interfering RNA was significantly suppressed at the same
concentration of 5-fluorouracil (P<0.05)
compared to untransfected cells. The results of this study showed that there
was the constitutively activated NF-kB
signaling pathway in the ESCC cell lines. RNA interference targeting at p65
increased the sensitivity of the ESCC cell lines to 5-fluorouracil, suggesting
that NF-kB might be a good target for
cancer treatment.
Key words NF-kB; RNA interference;
esophageal squamous cell carcinoma; IkBa; IkBa kinase
Esophageal cancer is one of the most frequently diagnosed
carcinomas, ranked as the sixth most common cause of death among all cancers in
the world [1], and is especially prevalent in China. Esophageal squamous cell
carcinoma (ESCC) is still one of the most aggressive squamous cell cancers with
poor prognosis and rapid progression [2]. Although therapy strategies have been
improved, the prognosis of patients with ESCC is still not positive. The
5-fluorouracil (5-FU) is frequently used in combination therapy of esophageal
cancer, but some patients have a poor response to 5-FU-based chemotherapy.
Obviously, a better understanding of the molecular mechanisms of ESCC helps to
refine therapy. Recently, much evidence has demonstrated that nuclear factor-kB (NF-kB/Rel) plays a
critical role in carcinogenesis. NF-kB is a transcription factor discovered by Sen
and Baltimore as a regulator of the expression of the k light chain of
immunoglobulins in B cells [3]. The NF-kB/Rel family is composed of
five subunits, p50/p105 (NF-kB1), p65 (RelA), C-Rel, P52/p100 (NF-kB2) and RelB, which can
form various homo- or heterodimers. The most studied form is a heterodimer of
the p50 and p65 subunits predominant in many types of cells [4].
In most normal cells, NF-kB is inactive by its tight association with
the cytoplasmic inhibition proteins, named inhibitors of NF-kB (IkB), belonging to
a gene family comprising IkBa, IkBb, IkBe, IkBg, Bcl-3, p100 and p105 [5]. A variety of extracellular stimulus
factors, such as inflammatory cytokines, growth factors, DNA damaging agents,
and bacterial and viral products, trigger a common signal transduction pathway
based on the phosphorylation, ubiquitination and proteasome-dependent
degradation of IkB to freely active NF-kB [6]. The released and activated NF-kB is rapidly
translocated to the nucleus and binds to the promoter region in the relevant
downstream genes to activate a series of transcriptional events. Thus, the
phosphorylation of IkBa is an indispensable step to activate the NF-kB signaling
pathway, which is catalyzed by an IkB kinase (IKK), a complex composed of IKKa, IKKb and IKKg. IKKb is the main
catalytic subunit of the IKK complex in the phosphorylation of IkBa at two
conserved serines (32 and 36) within the IkB N-terminal regulatory
domain [7].
NF-kB has a key function in the transformation, proliferation and
invasion of cancer cells as well as in resistance to radiotherapy and
chemotherapy. Constitutively activated NF-kB has been detected in many
human cancers including hepatocellular, colonic, pancreatic and cervical
cancers [8–11]. However, the high activation status of NF-kB and its
signaling pathway in ESCC has not been investigated. It has not been well
understood whether or not this pathway is responsible for the proliferation in
ESCC.
In this study, we investigated the mRNA and protein expression
levels of certain members of the NF-kB signaling pathway, as well as NF-kB activity and
the status of phosphorylation of IkBa in the two ESCC cell lines. The sensitivity
to 5-FU in ESCC cells transfected with or without small interfering RNA (siRNA)
targeting for p65 was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide assay. The results demonstrated that NF-kB was constitutively
activated in the ESCC cell lines with the phosphorylation of the IkBa protein and
overexpression of the genes. RNA interference (RNAi) targeting at p65 had
anti-proliferative effects and increased the sensitivity of the ESCC cells to
5-FU.
Materials and Methods
Cell culture
Two human ESCC cell lines, Eca109 and EC9706, were provided by the
State Key Laboratory of Molecular Oncology, Chinese Academy of Medical Science
(Beijing, China), and cervical cancer cell line HeLa229 as the positive control
cell was purchased from the Institute of Biochemistry and Cell Biology, Chinese
Academy of Sciences (Shanghai, China). Each of the three cell lines was
cultured in RPMI 1640 medium (Gibco-BRL, Rockville, USA) supplemented with 10%
fetal bovine serum (HyClone Laboratories, Logan, USA), 100 U/ml penicillin and
100 mg/ml streptomycin at 37 ºC with 5% CO2.
Antibodies and reagents
Mouse monoclonal antibodies to p65 (sc-8008), p-IkBa (sc-8404) and
IKKb
(sc-8014), and rabbit polyclonal antibodies to IkBa (sc-371) and p50 (sc-114)
were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). Biotin 3‘
end labeling kit (89818) and LightShift chemiluminescent electrophoretic
mobility shift assay (EMSA) kit (20148) were purchased from Pierce (Rockford,
USA). Avian myeloblastosis virus (AMV) first strand DNA synthesis kit (BS252),
Polymerase chain reaction (PCR) amplification kit (SK2491), oligonucleotides
and primers were obtained from Shanghai Sangon Biological Engineering &
Technology and Service (Shanghai, China). SignalSilence NF-kB p65 siRNA
(6261) was purchased from Cell Signaling Technology (Beverly, USA).
Immunocytochemical analysis
The immunoreactivity was determined using the SP kit according to
the manufacturer’s protocols. Briefly, the three cell lines were plated on
several glass slides and incubated at 37 ºC with 5% CO2 for 24
h. The slides were rinsed three times in phosphate-buffered saline (PBS, pH
7.4), and fixed with 4% formaldehyde at room temperature (RT) for 10 min. After
rinsing in PBS and treatment with 3% H2O2 for 10
min, the cells were blocked with 5% normal goat serum for 30 min in a humidified
box at RT to eliminate non-specific binding, then incubated with antihuman p50,
p65, IkBa and IKKb antibodies (1:100), p-IkBa (1:50), as well as PBS as the negative
control, at 4 ºC overnight. After the slides were rinsed three times in PBS and
incubated with corresponding secondary antibodies for 30 min, they were
developed with diaminobenzidine under a light microscope. Photomicrographs were
taken immediately (magnification, 400).
Preparation of cytoplasm and
nuclear proteins
Cytoplasm and nuclear proteins were extracted from the cells
cultured to approximately 90% confluence according to the instructions of the
nuclear and cytoplasmic extraction reagents kit (Pierce). Aliquots of the
proteins were stored at –70 ºC and the protein concentrations were determined by Bradford
method.
Western blot analysis
Cytoplasm (50 mg) from each cell line was added to 2´protein sample buffer, heated at 100 ºC for 5 min, and separated
using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE),
along with 20 ml of prestained protein molecular weight marker used as standard.
Proteins were electrotransferred to supported nitrocellulose membranes
(Amersham, Uppsala, Sweden) in transfer buffer containing 25 mM Tris, 193 mM
glycine, and 20% methanol. The membranes were blocked in 5% skimmed milk in
TBS-T (1´TBS, 0.05% Tween 20) at RT for 2 h,
then rinsed three times in TBS-T and incubated with anti-p50, anti-p65, anti-IkBa, anti-P-IkBa and anti-IKKb antibodies,
diluted in 1% skimmed milk, at RT for 2 h. The membranes were rinsed three
times in TBS-T and incubated with 1:5000 dilution of goat antirabbit or goat
antimouse secondary antibody conjugated to horseradish peroxidase for 1 h at
RT. After extensive washing with TBS-T, proteins bands on the membranes were
visualized by diaminobenzidine according to the manufacturer’s instructions.
Extracts (50 mg) from the nuclei were only detected in p50 and p65 proteins, as
mentioned above.
EMSA
To determine NF-kB activation, NF-kB oligonucleotide using biotin 3‘-end labeling was carried
out by EMSA. Briefly, the NF-kB oligonucleotide 5‘-AGTTGAGGGGACTTTCCCAGGC-3‘
DNA-binding sequence was labeled by the biotin
3‘ end labeling kit, and the nuclear proteins (10 mg) were
incubated for 20 min at RT with biotin-labeled DNA probes in the 20 ml of reaction
mixture comprising 25 mM EDTA, 5 mM MgCl2, 0.05%
NP-40, 2.5% glycerol, 50 mg/ml poly(dI∙dC) and 0.2 mg bovine serum albumin.
Nucleo-protein complexes were loaded onto the pre-electrophoresis 6%
non-denaturing polyacrylamide gels in 0.5´Tris-boric acid-EDTA buffer at 100 V for 2 h at RT. The
electrophoresed binding reactions were transferred to a nylon membrane
(Hybond-N+; Amersham) by capillary transfer system overnight at RT. The biotin
end-labeled DNA probe was detected using the conjugated
streptavidin-horseradish peroxidase and chemiluminescent substrate. The
membranes were exposed to -ray film for 2–5 min to obtain the perfect
signal. Another consensus sequence, OCT-1 5‘-TGTCGAATGCAAATCACTAGAA-3‘,
was used as the control for the quantity and quality of nuclear extracts.
RNA preparation and reverse
transcription-polymerase chain reaction (RT-PCR)
Total RNA was prepared from the ESCC cell lines with Trizol reagent
according to the manufacturer’s protocols and treated with the DNA-free kit to
remove residual genomic DNA. Briefly, the isolated RNA (1 mg) was reverse
transcribed to cDNA in a 20 ml reaction mixture containing 1 ml AMV reverse
transcriptase, 1 ml random hexamer, 4 ml 5´AMV buffer, 1 ml RNase inhibitor (20 U/ml), 2 ml dNTP (10 mM)
at 37 ºC for 1 h. The cDNA mixture (2 ml) was used for the PCR amplification mixture
(50 ml) containing 1.5 U Taq DNA polymerase in 10´PCR buffer, 1.5 mM MgCl2, 150 mM dNTP mixture,
and 50 pmol of sense and antisense primers.
Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was used as an internal
control. The following oligonucleotide primers were used for analysis of p65,
p50 and IkBa amplification by PCR. For p65 (630 bp), forward primer 5‘-ATAGAAGAGCAGCGTGGGGACT-3‘
and reverse primer 5‘-GGATGACGTAAAGGGATAGGGC-3‘; for p50 (449
bp), forward primer 5‘-AAACCTTTCCTCTACTATCCTGA-3‘ and reverse
primer 5‘-GCACTCTCTTCTTTTGTTCCTGT-3‘; for IkBa (316 bp),
forward primer 5‘-GAAGGAGCGGCTACTGGACG-3‘ and reverse primer 5‘-AATTTCTGTGTGGCTGGTTGGTGA-3‘;
and for GAPDH (915 bp), forward primer 5‘-AAGGTCGGAGTCAACGGATTTG-3‘
and reverse primer 5‘-CTTGACAAAGTGGTCGTTGAGG-3‘. PCR conditions:
4 min at 95 ºC for initial denaturing, followed by 30 cycles of 95 ºC for 1
min, annealing for 1 min at 55 ºC for p65, 60 ºC for p50 and 57 ºC for IkBa, then 72 ºC for
1 min and a final extension for 5 min at 72 ºC. The amplified products were
subjected to electrophoresis on 1% agarose gels.
RNAi
EC9706 (5´104 cells/well) and Eca109 (6´104 cells/well) cells were grown in six-well plates for 24 h in medium
without antibiotics before siRNA transfection. The cells were transfected for 5
h with 8 ml of siRNA (10 mM) using 5 ml of transfection reagents. Subsequently, 0.8 ml of normal growth
medium containing serum and antibodies was added to each well containing
transfected cells without removing the transfection mixture. After the
transfected cells were incubated for 72 h, they were collected to detect the
expression of p65 proteins by Western blot analysis.
Cell proliferation assay
To test the effect of RNAi targeting at p65, EC9706 and Eca109 cells
were transfected with p65 siRNA for 24 h in a 12-well plate. After the cells
were incubated for 24 h in a 96-well plate at a concentration of 1´104 cells per well, the cells were treated with
different concentrations of 5-FU for 24 or 48 h, respectively. The cells were
then incubated with 20 ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
solution (Sigma, St. Louis, USA) (at final concentration of 0.5 mg/ml) at 37 ºC
for 4 h. The medium was removed and the precipitated formazan was dissolved by
adding 200 ml dimethylsulfoxide (Sigma). After the samples were placed on a
shaker for 20–30 min, the absorbance at 570 nm was detected using a microplate
reader.
Statistical analysis
Data were analyzed by statistical software spss version 13.0 (SPSS, Chicago, USA). Comparisons between
the control and tested groups were performed using Student’s t-test.
Statistical results were expressed as means眘tandard deviations, except as otherwise stated. P<0.05 was
considered statistically significant.
Results
Expression of NF-kB subunits in cytoplasmic extracts of
ESCC cell lines
The two ESCC cell lines had intense immunoreactivity to p50 and p65
in the cytoplasm by immunocytochemistry [Fig. 1(A)]. Western blot
analysis of cytoplasmic extracts from the ESCC cell lines and the control cell
line (HeLa229) investigated the expression levels of p50 and p65. As shown in Fig.
1, the two ESCC cell lines had the same NF-kB activity in the cytoplasm
compared to the control cell line.
Expression and phosphorylation
status of IkBa
proteins
Because phosphorylation and degradation of IkBa are necessary for NF-kB activity, the
expression level of IkBa protein and its phosphorylation status were
detected by immunocytochemistry and Western blot to determine whether the high
NF-kB
activity in the two ESCC cell lines was caused by IkBa degradation. The results
in the two ESCC cells were compared with the control cells. The two ESCC cell
lines displayed different degree staining for IkBa and p-IkBa (Fig. 2). These results
demonstrated that IkBa proteins in the ESCC cell lines were phosphorylated, suggesting
that NF-kB activity was enhanced in the ESCC cells.
mRNA expression of p50, p65
and IkBa
in ESCC lines
The mRNA expressions of p50, p65 and IkBa in the two ESCC cell lines
were analyzed by RT-PCR. As shown in Fig. 3, the mRNA expressions of
p50, p65 and IkBa were detected in the ESCC cells. The results indicate that these
genes might play roles in the maintenance of constitutive NF-kB activity in
the two ESCC cell lines.
Increased NF-kB activity in ESCC lines
Western blot analysis of the nuclear extracts showed the same
expression levels of p50 and p65 in the ESCC lines compared with control cells
[Fig. 4(A)]. The NF-kB DNA-binding activity of nuclear protein extracts was analyzed by
EMSA with a double-stranded biotin-labeled oligonucleotide probe containing a
high-affinity kB sequence. The two ESCC cell lines showed a high level of NF-kB DNA-binding
activity [Fig. 4(B)].
Expression of IKKb proteins in ESCC cells
IkBa phosphorylation is mediated by IKK proteins, in the upstream of the
NF-kB
signaling pathway, therefore we examined the expression of IKKb by
immunocytochemical and Western blot analysis. IKKb proteins were expressed
and localized in the cytoplasm in the two ESCC cell lines (Fig. 5),
suggesting that the high levels of IKKb proteins might be another mechanism related
to the increased NF-kB binding activity in ESCC cells.
Expression of p65 transfected
with p65 siRNA
To examine whether p65 expression could be effectively inhibited by
siRNA after the cells had been transfected with p65 siRNA, the cells were
harvested after transfection for 72 h and the expression of p65 protein in the
cytoplasm was analyzed by Western blot. As shown in Fig. 6, the protein
level of p65 decreased after transfection with p65 siRNA, whereas the protein
level of MARK was not affected, suggesting that p65 siRNA can effectively
inhibit p65 protein levels.
Effects of 5-FU at different
concentrations combined with p65 siRNA on cell proliferation
To determine whether a combined effect exists between p65 siRNA and
5-FU, we compared the growth rates of the two ESCC cell lines with or without
transfection using siRNA at various of concentrations of 5-FU for 24 or 48 h.
As shown in Fig. 7, after exposure to 5-FU for 24 or 48 h, both cells
transfected with or without p65 siRNA were inhibited in a
concentration-dependent manner. But at the same concentration of 5-FU, cells
transfected with p65siRNA had a higher inhibitory effect than untransfected
cells. These results suggest that p65 siRNA increases the sensitivity of 5-FU
in the two ESCC cell lines.
Discussion
Aberrant activation of NF-kB has been shown in many cancers, such as
pancreatic, cervical, head and neck cancers [10–12], and non-Hodgkin’s
lymphoma [13]. Some reports have revealed that NF-kB is an important regulator
for several genes involved in the survival, transformation, differentiation,
invasion and growth of tumor cells [14–16]. Constitutively activated NF-kB has been
implicated in the carcinogenesis of those cancers. However, it is still unknown
whether the NF-kB signaling pathway is related to carcinogenesis of the esophagus.
This study demonstrated that the subunit of NF-kB in two ESCC cell lines,
which is composed of p50 and p65, the most common heterodimer of NF-kB, could bind to
DNA target sites. These results are consistent with the report published by
Morceau et al. [17]. Furthermore, the overexpression of p50 and p65
proteins was detected in both the cytoplasm and the nucleus in the ESCC cell
lines. The results of EMSA showed that NF-kB has high DNA binding
activity in the two ESCC cell lines. The activated heterodimer of p50 and p65
in the cytoplasm was translocated to the nucleus and regulated the targeted
genes. The NF-kB pathway was activated by a variety of stimuli, such as tumor
necrosis factor-a, interleukin-1, ionizing radiation and cancer chemotherapeutic
compounds. Without any possibility of inducing NF-kB activity in this study,
the high NF-kB activity is a constitutive and intrinsic feature of the two ESCC
cell lines. Due to the half-life of NF-kB being less than 30 min,
the maintenance of its activity needs ongoing protein synthesis [18].
Upregulated transcription factor activity is frequently caused by gene
overexpression [19]. In this study, the results of RT-PCR showed the
overexpression of p50 and p65 mRNA in two ESCC cell lines. The expression of
p50 and p65 was presumably a result of functional activation of NF-kB in the ESCC
cell lines. Therefore, activated high levels of NF-kB in the ESCC cell lines
mainly existed at translational, nuclear transporting and DNA-binding levels,
consistent with the results of Wang and Cassidy [20].
It is well known that degradation and phosphorylation of IkB, a natural
inhibitor of NF-kB in the cytoplasm, seems to be a critical reason to enhance NF-kB activation
[21]. IkBa is an important element of the IkB family. Previous reports
have identified that the cytoplasmic pool of IkB is degraded or
phosphorylated by different stimuli factors, which are dissociated from the NF-kB-IkB complex,
permitting the activated NF-kB to translocate to the nucleus and subsequently activate the
expression of some important target genes [22–24]. In this study, we
detected IkBa expression using immunocytochemistry, Western blot and RT-PCR. The
IkBa and p-IkBa proteins had
different expression levels in the cytoplasm of ESCC cell lines by
immunocytochemistry and Western blot analysis. The increased expression of IkBa mRNA in the ESCC
cell lines might be a feedback mechanism caused by the activated NF-kB, as the
activation of NF-kB is known to lead to the upregulation of IkBa as a feedback in other
cancers [25,26]. This suggests that upregulation of IkBa at the transcriptional
level and degradation of IkBa play an indispensable role in the constitutively activated NF-kB signaling
pathway.
The molecular basis of the activated NF-kB in many cancers is still
not known. However, a well-known signaling pathway including the activity of IkB-kinase, which
phosphorylates IkBa proteins, leads to its degradation [27,28]. IKKb, a
representative of molecule upstream of the activation NF-kB signaling
pathway, phosphorylates IkBa on serines 32 and 36 with approximately equal efficiency. By
immunocytochemistry and Western blot analysis, the expression of IKKb protein was
detected in the cytoplasm of the ESCC cell lines. Therefore, the overexpression
of IKKb might be one of the important mechanisms involved in the
constitutively activated NF-kB signaling pathway.
Activation of NF-kB seems to have many necessary functions, not only for tumor growth
but also for invasion and metastasis, as well as for inducing the resistance of
tumor cells to radiotherapy or chemotherapy [29–31]. Many studies using
different inhibitors affecting the activated IKKb/NF-kB pathway have
demonstrated that these methods have beneficial effects on tumor transformation
or increase sensitivity to radiotherapy or chemotherapy [32–34]. RNAi has
become a powerful strategy to knockdown and understand gene function. RNAi is a
general mechanism for the sequence-specific gene silencing induced by
double-stranded RNA [35]. RNAi is mediated by siRNA, a double-stranded RNA,
which is approximately 21–23 nucleotides and is specific for the sequence of its target [36].
In this study, it was demonstrated that the expression of p65 was
specifically inhibited by p65 siRNA. By combining siRNA targeting at p65 with
different concentrations of 5-FU, the two ESCC cell lines became sensitive to
5-FU at lower concentrations than those transfected cells without siRNA,
indicating that p65 siRNA with 5-FU treatment significantly inhibits cancer
cell growth. The results of this study demonstrate that the activated NF-kB signaling
pathway plays a crucial role in tumor cell growth, and suppression of p65 by
siRNA increases the sensitivity of the ESCC cells to 5-FU. The constitutively
activated NF-kB signaling pathway in the ESCC cell lines might also be an
important mechanism responsible for the survival and proliferation of the ESCC
cells. The amplification of p50, p65 and IkBa or the degradation of IkBa by IKKb might be the
main cause to influence the activity of NF-kB. Using the p65 siRNA, and
reducing NF-kB activity, the two ESCC cells show an enhanced sensitivity to anticancer
agents. Further studies are underway to investigate the action of the NF-kB signaling
pathway and to evaluate the potential use of the NF-kB signaling pathway as a
specific target for therapeutic strategies in ESCC with high NF-kB activity.
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